The response of cells to their environment is often mediated by soluble protein growth and differentiation factors. These factors exert their effects by binding to and activating transmembrane receptors. This interaction is the initial event in a cascade which culminates in a biological response by the cell. An important class of transmembrane receptors is the receptor protein tyrosine kinases (receptor PTKs, reviewed in van der Geer et al. Ann. Rev. Cell. Biol. 10, 251-337 (1994). PTKs consist of an extracellular domain which interacts specifically with the receptor's cognate ligand, a membrane spanning domain, and an intracellular domain which harbors the tyrosine kinase activity. Receptor PTKs are activated by ligand-mediated dimerization followed by autophosphorylation of tyrosine residues in the cytoplasmic domain. The receptor PTK can then in turn phosphorylate substrate molecules in the signal transduction pathway, leading to a cellular response.
The family of receptor PTKs can be divided into a number of sub-families based on the general structure of the extracellular domain and on amino acid sequence relationships within the catalytic domain. Currently, the largest known sub-family of receptor protein tyrosine kinases is the EPH-like receptors, consisting of at least 13 members. Members of this sub-family include the following: EPH (Hirai et al., Science 238, 1717-1725 (1987)), ECK (Lindberg et al., Mol. Cell. Biol. 10, 6316-6324 (1990)), Cek4, Cek5, Cek6, Cek7, Cek8, Cek9, Cek10 (Pasquale, Cell Regulation 2, 523-534 (1991); Sajjadi et al., The New Biologist 3, 769-778 (1991); Sajjadi and Pasquale Oncogene 8, 1807-1813 (1993)), Eek, Erk (Chan and Watt, Oncogene 6, 1057-1061 (1991)), Ehk1, Ehk2 (Maisonpierre et al., Oncogene 8, 3277-3288 (1993)), HEK (PCT Application No. W093/00425; Wicks et al., PNAS 89, 1611-1615 (1992)), HEK2 (Bohme et al., Oncogene 8, 2857-2862 (1993)), HEK5, HEK7, HEK8, HEK11 (U.S. Ser. No. 08/229,509) and HTK (Bennett et al. J. Biol. Chem. 269, 14211-14218 (1994)).
Until recently, no ligands for any member of the EPH sub-family had been identified. A ligand for the Eck receptor was described in PCT Application No. WO 94/11020 and Bartley et al. (Nature 368, 558-560 (1994)) and identified earlier as B61, a polypeptide encoded by a cDNA of unknown function (Holzman et al., Mol. Cell Biol. 10, 5830-5838 (1990)). Ligands for Elk and Ehk1 receptors have also been reported (PCT Application No. W094/11384; Davis et al., Science 266, 816-819 (1994)). Most recently, a polypeptide (ELF-1) identified from a mouse embryo midbrain and hindbrain cDNA library has been reported to be a ligand for Mek4 and Sek (Cheng and Flanagan, Cell 79, 157-168 (1994).
Most attempts to purify soluble factors from complex biological fluids have depended on cell-based bioassays of the response to stimulation by the factor. These include increased cell growth or survival, increased DNA synthesis, a chemotactic response, or some other downstream consequence of receptor activation. Receptor autophosphorylation has also been used as an assay to detect stimulation of the cell. We have recently described a method for the isolation of ligands based on direct detection of receptor/ligand binding and the use of receptor affinity chromatography for purification (Bartley et al., supra). Here we report the application of this method to purify, sequence, and molecularly clone one of a family of ligands corresponding to the EFH sub-family of receptor tyrosine kinases.
Although the EPH sub-family is the largest known sub-family of receptor PTKs, few ligands have been identified which bind to and activate an EPH sub-family receptor. It is therefore an objective to identify additional ligands for EPH sub-family receptor PTKs. These ligands will be useful for modulating responses of EPH sub-family receptor bearing cells.